WO2005078456A1

WO2005078456A1 - Method for detecting the formation of endothelins for medical diagnosis, and antibodies and kits for carrying out one such method

Info

Publication number: WO2005078456A1
Application number: PCT/EP2005/001359
Authority: WO
Inventors: Andreas Bergmann; Joachim Struck
Original assignee: B.R.A.H.M.S. Aktiengesellschaft
Priority date: 2004-02-13
Filing date: 2005-02-10
Publication date: 2005-08-25
Also published as: JP4685034B2; ES2255003T3; EP1564558A1; ATE312351T1; DE502004000540D1; US20080026414A1; US20120264149A1; US8124366B2; EP1564558B1; CN1918473A; US8450463B2; CN1918473B; HK1099807A1; JP2007522462A

Abstract

The invention relates to an in vitro method for detecting the formation of endothelins during serious illnesses, especially cardiovascular diseases, inflammations, sepsis and cancer, in the whole blood, plasma, or serum of a human patient, for medical diagnosis. According to said method, relatively long-lasting peptide fragments, especially a C-terminal peptide fragment, of the processed primary prepro-endothelins or pro-endothelins, that contain neither the actual biologically active endothelin nor the direct precursor thereof, big endothelin, are detected.

Description

Methods for determining the formation of endothelins for medical diagnostic purposes, and antibodies and kits for performing such a method

The invention relates to methods for determining the formation of endothelins in severe diseases by determining peptide fragments of the corresponding pro-endothelin, in particular a relatively long-lived C-terminal partial peptide of prepro-endothelin-1, in the circulation (whole blood, plasma or serum ) for the purpose of medical diagnostics, in particular in the context of sepsis diagnostics, cardiac diagnostics and e.g. also in cancer diagnosis and / or in general in the context of the diagnosis of such disease states in which endotheline play an important role in the course of the disease.

If the term "endothelin" is used in the context of the present application, this term stands primarily for endothelin-1 (ET-1). A corresponding statement, however, is often also valid for other isoforms of endothelins, which is why a restriction to endothelin-1 often does not appear to be necessary and the invention is also intended to extend to other endothelins in a broader sense. In this description, the term "diagnostics" is basically used as a simplifying generic term, which is also intended to include, in particular, prognostics / early prognostics and follow-up therapy monitoring.

The determinations are carried out in particular by means of specific immunodiagnostic methods, in particular by means of immunoassays of a type in which at least one labeled antibody is used (sandwich assay; co-operative assay, e.g. according to the SPALT or SPART principle).

Endothelin-1 (ET-1), a 21 amino acid peptide, is the most potent vasoconstrictor known. Since its discovery in 1988 by Yanagisawa and colleagues [27; Figures in square brackets refer to the attached literature list], biosynthesis, mode of action and association with diseases have been extensively investigated and summarized in current review articles [1, 1, 12, 17, 24]. There are three isoforms of endothelin (endothelin-1, endothelin-2, endothelin-3) encoded by different genes, of which endothelin-1 is present in the greatest concentrations and is the most effective. Endothelin-1 is synthesized in endothelial cells, in the lungs, in the heart, in the kidneys and in the brain. The primary translation product of the human endothelin-1 gene is a 212 amino acid peptide, prepro-endothelin-1 (SEQ ID NO: 1). In the secretion process, the signal peptidase removes a short N-terminal signal sequence (amino acids 1-17) of the prepro-endothelin. The resulting pro-endothelin is then processed by the protease furin on dibasic amino acid pairs to form a biologically inactive, 38-amino acid peptide big-endothelin (SEQ ID NO: 3), from which finally using endothelin-covering enzymes (ECEs) the mature, biologically active endothelin-1 (SEQ ID NO: 2) is formed. Endothelin works by binding to specific receptors that are localized on muscle cells, myocytes and fibroblasts. This bond leads to calcium efflux, activation of phospholipase C and inhibition of Na / K ATPase. In addition to the vasoconstrictive effect, endothelin also has growth-regulating properties.

In view of the detectable and suspected numerous and serious physiological effects of endothelins, especially endothelin-1, various assays for its immunodiagnostic determination have been developed since the time of its identification and used for measurements of endothelin (s), especially in human plasmas. The results of such determinations have been the subject of numerous publications.

Increased plasma concentrations of endothelin-1 and big-endothelin have been described for various clinical pictures [17]. These include cardiovascular diseases [1] (including pulmonary hypertension [21], atherosclerosis [13], congestive heart failure [25], heart attack [20]), sepsis and septic shock [11, 22, 23], cancer [2, 3 , 15, 18], among others.

The immunoassays used to measure endothelin in plasma samples (see the overview in [17]) belonged in particular to the type of radioimmunoassay (with labeled endothelin-1 as a competitor) or to the EIA / ELISA type and aimed exclusively at determining the endothelin from or on the determination of an endothelin immunoreactivity. RIA-type assays have a lower specificity and also detect related peptides containing the endothelin sequence.

However, it was found that endothelin-1 (ET-1) has an extremely short residence time in the circulation and that it is removed from the circulation after only 1-2 minutes [6]. Since endothelin-1 is considered stable in blood and plasma [6], the most important reason for the short residence time is its distribution in other tissues and its fast and viewed high affinity binding to receptors. As a result, significantly higher endothelin-1 concentrations could be determined in certain tissues and body fluids than, for example, in plasma [1, 7]. In view of these circumstances, the validity of the determination of ET-1 in plasma samples was questioned [17]. It can be assumed that the physiological effects of endothelin (ET-1) are not important for the instantaneous concentrations that can be determined in a plasma sample, which may only reflect a state of passage, but that the sum of all free and bound, eg tissue and receptor bound physiological ET-1 concentrations are of much greater relevance.

The determination of the ET-1 precursor, the so-called big endothelin ("big ET-1"; SEQ ID NO: 3) has the advantage over the determination of ET-1 that the residence time of "big-ET-1 "the circulation is significantly higher than that of the ET-1 released from it. In a series of investigations, this "big endothelin" was therefore determined instead of the actual endothelin. In particular, assays of the sandwich type were used for its specific determination, which enable a reliable differentiation of the big-endothelin-1 from the processed ET-1 and other endothelins [4, 8, 10]. They showed that in certain diseases the elevated ET immunoreactivities can be attributed to big ET.

The selective measurement of the big-ET-1 is only a gradual improvement, but not an actual solution to the problem, because big endothelin can also be quickly processed into endothelin in the blood circulation [1, 5, 9]. It also has a relatively short biological half-life (20-25 minutes) [10], and consequently a measured value for the big endothelin that can be determined in plasma also only represents an instantaneous plasma concentration and does not reflect the actually physiologically effective concentrations Endothelin. ET-1, which is physiologically formed under the conditions of a disease but is already processed and bound in tissues or to receptors, is not detected in the determination of big ET-1 in plasma. The total amount of physiologically active endothelin is therefore also underestimated when measuring big endothelin. Attempting a supplementary specific measurement of the C-terminal peptide fragment of big-ET-1 formed in the enzymatic cleavage of big-ET-1 in addition to ET-1 (with amino acids 74-90 of prepro-endothelin or amino acids 22-38 of big endothelin) showed that this peptide is even less stable than ET-1 and is therefore not suitable for measurements [10].

For the measurement of areas of the pro-endothelin outside of the big-endothelin, only one commercial competitive test is known from the prior art (N-terminal area 18-50, commercially available from Phoenix Pharmaceuticaluticals; application for sepsis diagnosis described in WO00 / 22439). No information is published on the stability and nature of the analyte to be measured with this assay.

The present invention has set itself the task of developing a determination method which enables the endogenous formation of big endothelin and endothelin, i.e. reflects the total physiological concentration and thus the effect of endothelin more reliably than the previous determinations of ET or big-ET in plasma.

Such a procedure should be valid and suitable for routine use and should be able to provide reliable values for the physiological production of ET (ET-1) and / or its precursors in various disease states, in particular in sepsis or other disease states in which increased values for endothelin play a role , This object is achieved in that ET or big-ET is not determined for diagnostic purposes in a whole blood, plasma or serum sample of a human patient, but rather a comparatively long-lived prepro or pro-endothelin partial peptide which contains the ET or does not contain big ET sequences, in particular a C-terminal partial peptide which at least contains the amino acids 168-212 of the pre-proET-1.

Claim 1 relates to the teaching of the present invention. Advantageous and currently preferred embodiments of the invention can be found in the subclaims.

The invention is based on experimental investigations by the applicant, in which it was able to show that those parts of the prepro-endothelin which are not direct precursors of the endothelin comprise long-lived peptides suitable for measurement purposes, which are measured reliably in blood samples and with a high clinical value can be.

Endothelin-1 is formed physiologically by processing the larger precursor molecule prepro-endothelin (SEQ ID NO: 1) or the secreted pro-endothelin obtained therefrom. In such a processing, in addition to big endothelin (and endothelin therefrom), further peptides have to be formed in primarily stoichiometric amounts, which, however, have never been the subject of scientific studies before and nothing is known about their possible further processing and stability. At the beginning of the investigations of the applicant there was hope that it could be shown that at least one of the hypothetical further peptide cleavage products would be found in blood samples (whole blood, plasma or serum samples) and would prove to be relatively stable and could therefore be suitable, as a measure of the physical logical formation of endothelines to serve regardless of a current endothelin concentration measurable in plasma.

The dimensioning of such a cleavage product could thus represent the desired method for determining the physiological endothelin concentration or production, which is referred to in the claims as determining the "formation of endothelin". This term refers to the fact that - assuming only one, namely the only known pathway of formation of endothelin-1 from prepro-endothelin - the physiological concentrations of endothelin-1 formed in connection with a disease only the amount of the previously processed Prepro-endothelins or pro-endothelins can correspond. If the partial peptides formed in addition to big endothelin or endothelin in the same stoichiometric concentration represent stable "metabolic slugs" that are neither bound to receptors nor distributed in tissues, they must be found in the circulation. Without necessarily wishing to imply a specific physiological mechanism, the "determination / measurement of the formation of endothelin" can also be regarded as a measurement of "secretory activity" or "secretory pro-endothelin production".

In this application, the peptide fragments to be determined are identified as "long-lived". By this term it is meant that the residence time of the peptide fragment to be determined in the circulation (in whole blood) is considerably longer than that of the endothelin or the big endothelin fragments. In particular, "long-lived" means that such peptide fragments in whole blood and a plasma obtained therefrom are not subject to any further rapid proteolytic cleavage and, compared to the rate of binding of the endothelin to receptors and the proteolytic cleavage of fragments, at a significantly slower rate from the Circulation or Metabolism to be removed.

Because of the longer stability or "longevity" mentioned, in the presence of such fragments, the information about the secretory activities which have already expired is stored for a period of time which is at least suitable for unproblematic measurement. Assuming, for example, that the endothelin precursors are released in the form of a single short-term release, the amount of "long-lived" fragments that can be measured after a certain time corresponds to the amount released originally, minus just an amount equal to the physiological half-life of the peptide fragment to be measured is linked in the circulation. On the other hand, assuming, for example, a more or less continuous production of the endothelin precursor during the course of the disease, the measurable concentration of a peptide fragment that is long-lived in the above sense reflects the previous physiological production of the precursor cumulatively, again only reduced by the concentration reduction that occurred in the same period of the peptide fragment according to its physiological clearance rate. The active endothelin, or its predecessor big-endothelin, can be processed or removed from the circulation long ago in the same period and, for example, bound to receptors and therefore no longer measurable. The longer a peptide fragment is long-lived, or the slower its clearance rate is, the less influence the measurement time has on the correctness of the determination of the above-mentioned "formation" of a biomarker, ie the endothelin. In this picture, a constant concentration over longer periods means that education and clearance are 'in balance'. If the concentration drops, this may indicate that the secretion of the precursor molecule (eg proendothelin) has ceased, for example because the molecular stores are exhausted, and the changes in concentration that can be observed are only determined by the clearance speed. The results of the measurement of a long-lived peptide fragment without a known physiological function thus provide both quantitatively and qualitatively different results than a measurement of a rather short-lived active peptide or its likewise relatively short-lived precursor.

The investigations of the applicant described in more detail below showed that the approach described above yields fruitful results in the determination of the formation of endothelins.

The investigations carried out and the most significant results of these investigations are explained in more detail below, reference being made to figures.

Show:

FIG. 1 shows a typical standard curve for the presently preferred sandwich assay with two antibodies, which is described in more detail in the experimental section and bind to amino acid sequences which correspond to positions 168-181 and 200-212 of prepro-endothelin-1, for determining a C-terminal pro Endothelin peptide sequence in human plasma;

FIG. 2 is a diagram which shows that when EDTA plasma samples from septic and cardiological patients are stored at room temperature for 12 hours, there is no significant loss of immunoreactivity in an assay according to FIG. 1;

FIG. 3a shows the measurement of plasmas from 5 groups of human patients with various diseases / diagnoses, compared with the measurements for apparently healthy people; the dotted line shows the maximum value found in healthy people (line for 100% specificity related to healthy Controls);

FIG. 3b shows a representation corresponding to FIG. 3a, for four further groups of patient plasmas;

In its most general aspect, the method according to the invention relates to the determination of a relatively long-lived peptide fragment of pro-endothelin-1, which does not contain the amino acid sequences of endothelin-1 or its precursor big-endothelin, in whole blood, plasma or serum samples, ie in the circulation of patients, for the indirect determination of the formation of endothelins, in particular endothelin-1, in the case of serious diseases. In a preferred embodiment, the particular peptide fragment is a C-terminal fragment to which two antibodies bind that bind to peptides with amino acid sequences corresponding to positions 168-181 and 200-212 of prepro-endothelin-1.

For the practical implementation of the invention, non-competitive sandwich assays, e.g. the type, - = w-is - e-r-was used for in-depth in-depth examinations and is described in more detail below.

Non-competitive sandwich immunoassays (two-sided immunoassays) have a number of advantages over competitive immunoassays, including the fact that they can be designed better than solid-phase assays (heterogeneous assays), can be more robust in terms of manageability, and measurement results with a higher one Can deliver sensitivity and are also better suited for automation and series measurement. In addition, in comparison with competitive immunoassays that work with only one type of antibody, they can also provide additional information in that sandwich immunoassays recognize only those molecules or peptides in which both binding sites for the antibodies used for sandwich formation are on the same molecule available. The antibodies which can be used can in principle be any suitable monoclonal and / or polyclonal antibodies, although polyclonal antibodies which have been purified from affinity are currently preferred.

The antibodies are particularly preferably obtained by immunizing an animal, in particular sheep, with an antigen which contains a synthetic peptide sequence which corresponds to a short amino acid sequence of prepro-endothelin-1 and has an additional cysteine residue at the N-terminus. The following experimental section describes in particular antibodies or their use in an assay, which bind to the amino acid sequences 161-181 and 200-212. However, additional antibodies were also used in the course of the investigations, which bind to positions 184-203 and 136-148 accordingly. The supplementary results obtained with these additional antibodies during measurements are only discussed in general terms in this application.

In a preferred embodiment, the method is carried out as a heterogeneous sandwich immunoassay, in which one of the antibodies is attached to any solid phase, for example the walls of coated test tubes (for example made of polystyrene, coated tubes; CT) or on microtiter plates, for example made of polystyrene , or is immobilized on particles, for example magnetic particles, while the other antibody carries a residue which is a directly detectable label or enables a selective link to a label and serves to detect the sandwich structures formed. A delayed or subsequent immobilization using suitable solid phases is also possible.

In principle, all labeling techniques that can be used in assays of the type described can be used, including labels with radioisotopes, enzymes, fluorescence, chemiluminescence or bioluminescence labels and directly optically detectable color markings, such as gold atoms and dye particles, such as are used in particular for so-called point-of-care (POC) or rapid tests for determination in whole blood samples. In the case of heterogeneous sandwich immunoassays, the two antibodies can also have parts of a detection system of the type described below in connection with homogeneous assays.

It is therefore within the scope of the present invention to also design the method according to the invention as a rapid test.

The method according to the invention can also be designed as a homogeneous method in which the sandwich complexes formed from the two antibodies and the peptide fragment to be detected remain suspended in the liquid phase. In such a case, it is preferred to label both antibodies with parts of a detection system which, when both antibodies are integrated in a single sandwich, enables signal generation or triggering. Such techniques can be designed in particular as fluorescence amplification or quenching detection methods. A particularly preferred method of this type relates to the use of detection reagents to be used in pairs, as described, for example, in US Pat. No. 4,822,733, EP-Bl-180 492 or EP-Bl-539 477 and the prior art cited therein. They enable a measurement that selectively only detects reaction products that contain both labeling components in a single immune complex, directly in the reaction mixture. As an example, reference should be made to the technology offered under the brands TRACE ^® (Time Resolved Amplified Cryptate Emission) and KRYPTOR ^® , which implements the teachings of the above registrations.

It was shown in the investigations by the applicant that the determination according to the invention of the C-terminal peptide fragment of prepro-endothelin-1 was highly interesting and delivers relevant measurement results. As shown below, this statement applies not only to sepsis diagnosis, but also to cardiac diagnosis and cancer diagnosis.

It is further assumed that the determination method according to the invention can also be carried out particularly advantageously in the context of a so-called multiparameter diagnosis, both in the field of cardiac diagnosis as well as sepsis and cancer diagnosis. Other parameters determined here are, for example, the cardinal parameters ANP, BNP, proANP, proADM or proBNP or sepsis parameters, which e.g. are selected from the group consisting of anti-ganglioside antibodies, the proteins procalcitonin, CA 125, CA 19-9, S100B, SlOOA proteins, LASP-1, soluble cytokeratin fragments, in particular CYFRA 21, TPS and / or soluble cytokeratin 1 fragments (sCYlF), the peptides inflammin and CHP, other peptide prohormones, glycine-N-acyltransferase (GNAT), carbamoyl phosphate synthetase 1 (CPS 1) and the C-reactive protein (CRP) or fragments from that. The multiparameter determinations mentioned provide for the measurement results for several parameters to be determined simultaneously or in parallel, and e.g. with the help of a computer program that also uses diagnostically significant parameter correlations.

The invention will hereinafter be described by a description of the preparation of the preferred assay components, the implementation of a preferred embodiment of a sandwich-type assay and the results obtained by using such an assay, the determination of a C-termian peptide fragment in EDTA plasmas from control persons, and of sepsis, heart and cancer patients explained in more detail.

Experimental part

A. Materials and methods 1. Peptide Syntheses

Derived from the known amino acid sequence of human prepro-endothelin-1 (SEQ ID NO: 1), three areas were selected (items 168-181, 184-203, 200-212). Complemented by an N-terminal cysteine residue, these areas were chemically synthesized as soluble peptides using standard methods, purified, quality-controlled using mass spectrometry and reversed phase HPLC and lyophilized in aliquots (JERINI AG, Berlin, Germany). The amino acid sequences of the peptides are:

Peptide PCT15 (168-181 + N-terminal cysteine)

CRSSEEHLRQTRSET (SEQ ID NO: 4)

Peptide PCW14 (200-212, + N-terminal cysteine)

CSRERYVTHNRAHW (SEQ ID NO: 5)

Peptide PNR20 (184-203; + N-terminal cysteine)

NSVKSSFHDPKLKGKPSRER (SEQ ID NO: 6)

The following peptide was also synthesized as the standard for calibrating the assays:

Standard peptide PSW44 (169-212) SSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAHW (SEQ ID NO: 7)

2. Conjugation and immunization

The MBT (m-maleimidobenzoyl-N-hydroxysuccinimide ester) was used to conjugate the peptides PCT15 and PCW14 to the carrier protein KLH (keyhole limpet hemocyanin) (see operating instructions "NHS-Esters-Maleimide Crosslinkers", PIERCE, Rockford, IL, USA) , Sheep were immunized with these conjugates according to the following scheme: Each sheep was initially given 100 μ <_ conjugate (mass specification based on the peptide content of the conjugate) and then 4 μl conjugate each 4 weeks (Mass data based on the peptide content of the conjugate). Starting with the fourth month after the start of the immunization, 700 ml of blood were drawn from each sheep every 4 weeks and antiserum was obtained therefrom by centrifugation. Conjugations, immunizations and antisera extraction were carried out by MicroPharm, Carmarthenshire, UK.

3. Purification of the antibodies

In a 1-step procedure, the peptide-specific antibodies were prepared from the antisera which had been obtained beginning with the fourth month after the immunization.

For this purpose, the peptides PCT15 and PCW14 were first coupled to SulfoLink Gel (see instructions for use "SulfoLink Kit" from PIERCE, Rockford, IL, USA). 5 mg of peptide per 5 ml of gel were offered for coupling.

The affinity purification of peptide-specific antibodies from sheep antisera against both peptides was carried out as follows:

The peptide columns were first washed three times alternately with 10 ml elution buffer r (50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate, 0.1% Tween, pH 6.8). 100 ml of the antisera were filtered through 0.2 μm and the existing column material was added. For this purpose, the gel was rinsed out of the column quantitatively with 10 ml of binding buffer. The incubation was carried out overnight at room temperature with swirling. The batches were transferred quantitatively into empty columns (NAP 25, Pharmacia, emptied). The runs were discarded. The mixture was then washed protein-free with 250 ml of binding buffer (protein content of the wash eluate <0.02 A280 nm). Elution buffer was added to the washed columns and 1 ml fractions became collected. The protein content of each fraction was determined using the BCA method (see work instructions from PIERCE, Rockford, IL, USA). Fractions with protein concentrations> 0.8 mg / ml were pooled. After protein determination of the pools using the BCA method, yields of 97 mg were found for the anti-PCT15 antibody 0407-pAk and 60 mg for the anti-PCW14 0410-pAk antibody.

4. Marking

The anti-PCW14 0410-pAk antibody was treated as follows:

Using a NAP-5 gel filtration column (Pharmacia), 500 μl of the purified antibody was buffered in 1 ml of 100 mM potassium - phosphate buffer (pH 8.0) according to the instructions. The protein concentration determination of the antibody solution gave a value of 1.5 mg / ml.

For the chemiluminescent labeling of the antibody, 67 μl of the antibody solution were mixed with 10 μl MA70 acridinium NHS ester (1 mg / ml; HOECHST Behring) and incubated for 15 minutes at room temperature. Then 423 μl of 1 M glycine were added and incubated for a further 10 minutes. The labeling approach was then buffered in a 1 ml mobile phase A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4) using a NAP-5 gel filtration column (Pharmacia) according to the work instructions and thereby freed from low-molecular components. A gel filtration HPLC (column: Waters Protein Pak SW300) was carried out to remove the last residues of labels not bound to antibodies. The sample was applied and chromatographed with solvent A at a flow rate of 1 ml / min. The wavelengths 280 nm and 368 nm were measured with a flow photometer. The absorption ratio 368 nm / 280 nm as a measure of the degree of labeling of the antibody was 0.10 at the peak. The fractions containing monomeric antibodies

(Retention time 8-10 min) were collected and in 3 ml of 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% sodium azide, pH 7.4.

5. Coupling

The anti-PCT15 antibody 0407-pAk was treated as follows:

Irradiated 5 ml polystyrene tubes (from Greiner) were coated with purified antibody as follows: The antibody was dissolved in 50 mM Tris, 100 mM NaCl, pH 7.8 at a concentration of 6. Diluted 6 μg / ml. 300 μl of this solution were pipetted into each tube. The tubes were incubated at 22 ° C for 20 hours. The solution was suctioned off. Then each tube was filled with 4.2 ml of 10 mM sodium phosphate, 2% Karion FP, 0.3% Bovine Serum Albumin, pH 6.5. After 20 hours the solution was suctioned off. Finally, the tubes were dried in a vacuum dryer.

B. Execution and evaluation of the immunoassay

An assay buffer of the following composition was prepared: 100 mM sodium phospha, 150 mM NaCl, 5% bovine serum albumin (BSA), 0.1% non-specific sheep IgG, 0.1% sodium azide, pH 7.4

The above material served as the standard material. chemically synthesized peptide (peptide PSW44) corresponding to positions 169-212 of pre-pro-endothelin-1. This was serially diluted in horse normal serum (company SIGMA). Concentrations according to the weight of peptide were ascribed to the standards produced in this way.

Measurement samples were EDTA plasmas from apparently healthy people, from patients with sepsis and from patients with various cardiovascular diseases. 50 μl standards or samples and 200 μl assay buffer were pipetted into the test tubes. It was incubated for two hours at 22 ° C. with shaking. The mixture was then washed 4 times with 1 ml of washing solution (0.1% Tween 20) per tube and allowed to drain. Then 200 μl assay buffer containing 1 million RLU (relative light units) of the MA70-labeled antibody were pipetted. It was incubated for two hours at 22 ° C. with shaking. The mixture was then washed 4 times with 1 ml of washing solution (0.1% Tween 20) per tube, drained and the chemiluminescence bound to the tube was measured in a luminometer (company BERTHOLD, LB952T; Basisreagenzien BRAHMS AG).

Using the MultiCalc (Spline Fit) software, the concentrations of the samples were read from the standard curve.

C. Results

The analyte that can be measured with the developed sandwich immunoassay (antibodies against positions 168-181 and 200-212) is referred to below as C-terminal pro-endothelin or Ct-pro-endothelin. A typical standard curve for the developed test is shown in FIG. 1. With the test, Ct-pro-endothelin concentrations of well below 50 pg / ml could still be determined.

To examine the question of whether problems due to insufficient stability in a sample or measurement solution must be expected when measuring the C-terminal peptide fragment, 5 sepsis plasmas were measured fresh and after 12 hours of storage at room temperature. The results are summarized in Figure 2. They show that after 12 days of storage, the immunoreactivity was almost unchanged at about 93% of the initially measured immunoreactivity. This proven stability is a great advantage from a handling point of view for diagnostics The test was used to measure plasmas from cardiological and septic patients. The results obtained are shown in Figures 3a and 3b. For all cardiological clinical pictures examined, higher values were found compared to normal controls. Increased values were also found for patients with SIRS (systemic inflammatory response syndrome) and septic conditions. The diagnostic sensitivity (given 100% specificity based on healthy controls) increased with the severity of the disease: sepsis 32.3%, severe sepsis 65.5%, and septic shock 75%.

The samples were measured with a modified assay in which one of the antibodies of the above. Sandwich assays were replaced by an antibody that recognized amino acids 184-203 of prepro-endothelin-1, as expected, essentially identical results were obtained.

On the other hand, if one of the antibodies used recognized an amino acid sequence that is located closer to the N-terminus of prepro-endothelin (32-52 or 136-148), no higher measured values could be obtained compared to healthy individuals. This indicates that pro-endothelin as such was not present in the measured plasma samples and is not only processed proteolytically with the formation of big-endothelin, but also that the C-terminal sequence 93-212 set in the process is still further is cleaved, at least one such cleavage site must be in the region of amino acids 149-167. This statement applies to the plasmas of patients with the diseases examined. However, it cannot be ruled out that in other patient groups, for example, the entire C-terminal fragment 93-212 is preserved and its selective measurement can deliver diagnostically relevant results. Literature list file 4295

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Claims

claims

1. In vitro method for determining the formation of endothelins in severe diseases, in particular cardiovascular diseases, inflammation, sepsis and cancer, in whole blood, plasma or serum of a human patient for the purposes of medical diagnosis, characterized in that the Formation of endothelin-1 (SEQ ID NO: 2) and big-endothelin-1 (SEQ ID NO: 3) by determining such C-terminal fragments of prepro-endothelin-1 (SEQ ID NO: 1), which are recognized by antibodies which bind to peptides which correspond to peptide sequences in the region of amino acids 93 to 212 of prepro-endothelin-1.

2. The method according to claim 1, characterized in that the determination in the biological liquid is carried out with the aid of an immunoassay which works with at least one labeled antibody which specifically recognizes only the peptide fragment to be determined.

3. The method according to claim 2, characterized in that the immunoassay is a competitive immunoassay or a sandwich immunoassay.

4. The method according to claim 1, characterized in that one determines those C-terminal fragments of prepro-endothelin-1 that are recognized by antibodies that bind to peptides, the peptide sequences in the range of amino acids 168 to 212 (SEQ ID NO: 7) correspond to prepro-endothelin-1.

5. The method according to claim 4, characterized in that for the determination of a C-terminal fragment with amino acids 168 to 212 (SEQ ID NO: 7) of prepro-endothelin-1 pairs of antibodies are used which are linked to two different peptide sequences bind, which are selected from peptide sequences with the amino acids 168-181, 184-203 and 200-212 of prepro-endothelin-1.

6. The method according to any one of the preceding claims, characterized in that it is a method for the quantitative or for the semi-quantitative determination of the peptide fragments to be determined.

7. The method according to claim 6, characterized in that it is an immunochromatographic point-of-care test or another rapid test.

8. The method according to any one of claims 4 to 7, characterized in that the antibodies used for the determination are monoclonal and / or affinity-purified polyclonal antibodies.

9. The method according to any one of claims 4 to 8, characterized in that antibodies are used which have been obtained by immunizing an animal with an antigen which contains a synthetic peptide selected from the peptides (SEQ ID NO: 4), (SEQ ID NO: 5) and (SEQ ID NO: 6).

10. The method according to any one of claims 4 to 9, characterized in that two antibodies are used for the determination, one of which is labeled and the other is bound to a solid phase or can be selectively bound to a solid phase.

11. The method according to any one of claims 4 to 9, characterized in that two antibodies are used for the determination, both of which are dispersed in the liquid reaction mixture, a first labeling component being bound to the first antibody and being part of a fluorescent or labeling system based on chemiluminescence deletion or amplification, and the second labeling component of this labeling system is bound to the second antibody, so that after binding of both antibodies a measurable signal is generated on the peptide fragment to be detected, which enables detection of the sandwich complexes formed in the measurement solution.

12. The method according to claim 11, characterized in that the marking system comprises rare earth crypates or chelates in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

13. The method according to any one of claims 1 to 123, characterized in that it is used for diagnosis, for the determination of the severity and for the prognosis as well as for the therapy control of sepsis during the course of treatment.

14. The method according to claim 13, characterized in that it is carried out as part of a multi-parameter determination, in which at least one further parameter relevant for sepsis diagnosis is determined at the same time.

15. The method according to claim 14, characterized in that the further parameter (s) relevant for sepsis diagnosis are selected from the group consisting of anti-ganglioside antibodies, the proteins procalcitonin, CA 125, CA 19 -9, S100B, SlOOA proteins, LASP-1, soluble cytokeratin fragments, in particular CYFRA 21, TPS and / or soluble cytokeratin 1 fragments (sCYlF), the peptides inflammin and CHP, fragments of the prohormones pro-ANP, pro -BNP or pro-ADM, the glycine-N-acyltransferase

(GNAT), the carbamoyl phosphate synthetase 1 (CPS 1) and the C-reactive protein (CRP) or fragments thereof.

16. The method according to any one of claims 1 to 12, characterized in that it is applied in the field of cardiac diagnostics.

17. The method according to claim 16, characterized in that it is carried out as part of a multiparameter determination. in which other parameters relevant to cardiac diagnostics are determined at the same time.

18. The method according to any one of claims 1 to 12, characterized in that it is applied in the field of cancer diagnosis.

19. The method according to claim 18, characterized in that it is carried out in the context of a multi-parameter determination, in which at the same time further parameters relevant for cancer diagnosis are determined.

20. Antibodies that specifically bind to peptides consisting of the amino acid sequences corresponding to amino acids 168-181, 184-203 and 200-212 of prepro-endothelin-1.

21. Antibody according to claim 20, characterized in that they are affinity-purified polyclonal antibodies or monoclonal antibodies.

22. Kit for carrying out a method according to one of claims 1 to 19, characterized in that it comprises at least: (a) a first antibody according to one of claims 20 and 21, (b) a second, other antibody according to one of claims 20 and 21, one of the antibodies being labeled and the other immobilized or immobilizable, and (c) a standard peptide having an amino acid sequence comprising at least amino acids 168-203 or 168-212 of prepro-endothelin.

23. Kit according to claim 22, characterized in that the immobilized antibody is immobilized on the walls of a test tube (CT).